A central challenge for research in regenerative medicine is to develop cell compositions that can help reconstitute cardiac function. It is estimated that nearly one in five men and women have some form of cardiovascular disease (National Health and Nutrition Examination Survey III, 1988-94, Center of Disease Control and the American Heart Association). Widespread conditions include coronary heart disease (5% of the population), congenital cardiovascular defects (0.5%), and congestive heart failure (3%). The pharmaceutical arts have produced small molecule drugs and biological compounds that can help limit the damage that occurs as a result of heart disease, but there is nothing commercially available to help regenerate the damaged tissue.
With the objective of developing a cell population capable of cardiac regeneration, research has been conducted on several different fronts. Clinical trials are underway at several centers to test the use of autologous bone marrow derived cells for therapy after myocardial infarction (Perin et al., Circulation 107:2294, 2003; Strauer et al., Circulation 106:1913, 2002; Zeiher et al., Circulation 106:3009, 2002; Tse et al., Lancet 361:47, 2003; Stamm et al., Lancet 3661:45, 2003). It has been hypothesized that the cells may have a cleansing function to improve blood perfusion of the heart tissue. Clinical trials are also underway to test the use of autologous skeletal muscle myoblasts for heart therapy (Menasche et al., J. Am. Coll. Cardiol, 41:1078, 2003; Pagani et al., J. Am. Coll. Cardiol. 41:879, 2003; Hagege et al., Lancet 361:491, 2003). However, it is unclear if the contraction of striatal muscle cells can coordinate adequately with cardiac rhythm.
A more direct approach would be to use cells that are already committed to be functional cardiomyocytes. Syngeneic neonatal or postnatal cardiac cells have been used in animal models to repair damage resulting from permanent coronary occlusion (Reffelmann et al., J. Mol. Cell Cardiol. 35:607, 2003; Yao et al., J. Molec. Cell. Cardiol. 35:607, 2003. Accordingly, if such cells were available for human therapy, they could be very effective for the treatment of ischemic heart disease.
International patent publication WO 99/49015 (Zymogenetics) proposes the isolation of a nonadherent pluripotent cardiac-derived human stem cell. Heart cells are suspended, centrifuged on a density gradient, cultured, and tested for cardiac-specific markers. Upon proliferation and differentiation, the claimed cell line produces fibroblasts, muscle cells, cardiomyocytes, keratinocytes, osteoblasts, or chondrocytes. However, it is unclear whether any of the cell preparations exemplified in these publications can be produced in sufficient quantities for mass marketing as a therapeutic composition for regenerating cardiac function.
A potential source of regenerative cells for treating cardiac disease is pluripotent stem cells of various kinds, especially embryonic stem cells. Several laboratories have reported results using mouse ES cells (Wobus et al., J. Mol. Cell Cardiol. 29:1525, 1997; Kolossov et al., J. Cell Biol. 143:2045, 1998; Narita et al., Development 122:3755, 1996; L. Field, U.S. Pat. No. 6,015,671; Klug et al., J. Clin. Invest. 98:216, 1996; Doevendans et al., J. Mol Cell Cardiol. 32:839, 2000; Muller et al., FASEB J. 14:2540, 2000; Gryschenko et al., Pflugers Arch. 439:798, 2000).
Thomson et al. (Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully culture embryonic stem cells from primates, using rhesus monkeys and marmosets as a model. They subsequently derived human embryonic stem (hES) cell lines from human blastocysts (Science 282:114, 1998). Human embryonic stem cells can proliferate in vitro without differentiating; they retain a normal karyotype, and the capacity to differentiate to produce a variety of adult cell types.
However, a number of obstacles have stood in the way of developing a paradigm for obtaining substantially enriched populations of cardiomyocyte lineage cells from primate pluripotent stem (pPS) cells. Some ensue from the relative fragility of pluripotent cells of primate origin, the difficulty in culturing them, and their exquisite sensitivity and dependence on various factors present in the culture environment. Other obstacles ensue from the understanding that cardiac progenitor cells require visceral embryonic endoderm and primitive streak for terminal differentiation (Arai et al., Dev. Dynamics 210:344, 1997). In order to differentiate pPS cells into cardiac progenitor cells in vitro, it is necessary to mimic or substitute for all the events that occur in the natural ontogeny of such cells in the developing fetus.
Small patches of beating cells can be generated from hES cells by a generalized differentiation protocol, and it has recently been proposed that these cells be used for determining the effect of small molecule drugs of cardiomyocyte transmembrane potentials (WO 04/011603; Thomson, Kamp et al.). It has been proposed that differentiated cell populations containing a few cardiac cells can be generated simply by culturing in a medium supplemented with serum, and then somehow sorting out the beating cells (WO 04/081205; ES Cell International). It is unclear how cell populations having a low frequency of cardiomyocyte lineage cells can be used to generate preparations sufficiently pure for therapeutic use in a commercially viable manner.
Geron Corporation has developed novel tissue culture environments that allow for continuous proliferation of human pluripotent stem cells in an environment essentially free of feeder cells (see U.S. Pat. No. 6,800,480; Australian patent AU 751321, and International Patent Publication WO 03/020920). Feeder-free pPS cell cultures can be used to make differentiated cell populations free of xenogeneic contaminants, such as hepatocytes (U.S. Pat. No. 6,458,589), neural cells (U.S. Pat. No. 6,833,269), and cardiomyocytes (WO 01/88104).
Commercialization of these technologies for use in regenerative medicine will benefit from further improvement in the expansion and differentiation protocols to improve cell homogeneity and yield.